The proposed project will elucidate the mechanisms that control polar adhesin production, biofilm formation, and host colonization in alpha-proteobacteria, a physiologically heterogeneous group that includes a number of significant human pathogens. Particularly relevant to public health, biofilm formation plays a crucial role in the survival of bacteria in diverse environments: cells in biofilms attach recalcitrantly to biotic and abiotic surfaces, develop increased resistance to antimicrobial agents, and contribute to persistent infections. Although limiting biofilm formation has the potential to prevent and restrict microbial diseases, little is known about biofilm formation by alpha-proteobacteria and how it facilitates host colonization. We previously discovered a biofilm-associated mutation common in laboratory strains of Sinorhizobium meliloti, a model alpha-proteobacterium that forms mutualistic symbiosis with compatible legumes by colonizing root tissues and fixing nitrogen in exchange for nutrients from plant hosts. Correcting the mutation restored full-length production of the conserved polarity factor PodJ, thus enabling synthesis of the holdfast (a polar adhesin) and assembly of robust biofilms, phenotypes never observed before. We found that biofilm- competent strains possess a competitive advantage over biofilm-deficient strains during host infection. Via transposon mutagenesis, we identified a number of genes involved in biofilm development, including one encoding a conserved transcription factor (LdtR) and another encoding a diguanylate cyclase and phosphodiesterase, known to modulate levels of the c-di-GMP second messenger. The goals of this proposal are (a) to decipher the responsibilities of key regulators during biofilm formation and host colonization and (b) to establish a system for monitoring the relationship between symbiosis and environmental factors. We plan to achieve these goals by accomplishing the following three specific aims. (1) We will determine how LdtR expression is regulated and what cellular functions it performs. (2) We will assess how factors that modulate c- di-GMP levels contribute to biofilm formation and host infection. (3) We will evaluate whether symbiosis affects host response to toxic compounds and how chemical stress and other microbes influence host-symbiont interactions. Results from the investigation will provide a better model of how cellular and external factors can contribute to adhesion, biofilm formation, and host invasion in alpha-proteobacteria. In addition to accomplishing the scientific objectives described above, funding of this proposal will enhance the research productivity and grant competitiveness of the investigator and allow students, particularly those from underrepresented backgrounds, to gain research training and preparation for biomedical careers.